Delphinidin-3-rutino side relaxes the bovine ciliary smooth muscle through activation of ETB receptor and NO/cGMP pathway

Delphinidin-3-rutinoside (D3R) is the major anthocyanin component in blackcurrant (Ribes nigrum L.) fruits. We investigated the relaxation mechanism of D3R in bovine ciliary smooth muscle (CM). D3R at a concentration of 10(-5) M produced a sustained and progressive relaxation during the contraction induced by endothelin (ET)-1 in the bovine CM specimens. After the pre-treatment with D3R, the anthocyanin exerted an inhibitory effect on the ET-1-induced contraction with a concomitant increase in cyclic GMP production and decreased phosphorylation ratio of myosin light chain (RLC). The inhibitory effect of D3R was significantly attenuated in the presence of either N-G-nitro-L-arginine (NOARG) as a nitric oxide synthase (NOS) inhibitor, carboxy-PTIO as a NO scavenger, ODQ as an inhibitor of guanylyl cyclase, or BQ788 as a selective ETB receptor antagonist. The atteuation with NOARG was reversed by the addition of excess L-arginine. However, iberiotoxin as a Ca2+-activated K+ channel inhibitor, propranolol as a beta-adrenoceptor antagonist, and indomethacin as a cyclooxygenase inhibitor failed to modify the inhibitory effect of D3R. Scatchard plot analysis revealed that the [I-125]-ET-1 binding site constituted a single population with Kd of 54.5 +/- 4.6 nM and maximum binding site (B-max) of 168.4 +/- 25.4 fmol/mg protein in the ciliary epithelium (CE), and Kd of 141.7 +/- 18.0 nM and B-max of 357.7 +/- 35.8 fmol/mg protein in CM. [I-125]-ET-1 binding was completely displaced by BQ788 with K-i values of 56.7 +/- 10.8 pM in CE and 93.4 +/- 23.3 pM in CM. Meanwhile, partial displacement (approximately 40%) was observed by BQ123 as a selective ETA receptor antagonist in both preparations. ETB receptor was predominant subtype in CE and CM, whereas kinetics of the binding was different in two preparations. These results suggest that D3R possibly stimulates ETB receptors to produce/release NO, and results in an inhibition of myosin RLC phosphorylation and/or acceleration of dephosphorylation, thereby causing relaxation and producing an inhibitory effect on the ET-1-induced contraction in the bovine CM. (c) 2004 Elsevier Ltd. All rights reserved.